Equipment, Consumables and Solvents

Equipment

Consumables

Solvents

Standards and Buffers

  • Food Resuspension and TissueLyser buffer: 4:1 Methanol:Water
  • 6 mixes of standards at 1 ng/uL concentration from Prenni Lab
  • Crash solvent: 1% formic acid in 5:1 acetonitrile:water

Protocols

Homogenization and Extraction with TissueLyser LT Bead Beater Protocol

  1. Pre-chill the homogenization tubes, metal homogenization balls, and spatula on dry ice and/or liquid nitrogen.
  2. Remove lyophilized foods from freezer and place on dry ice and/or liquid nitrogen.
  3. Weigh cooled microfuge tube and ball on analytical balance and record or tare its mass.
  4. Transfer between 40-60 mg of lyophilized food using a cooled spatula to cooled microfuge tube and record its mass.
  5. Add 12uL of room temperature 4:1 Methanol:Water per 1mg lyophilized food.(e.g. 600 uL for 50 mg of food)
    Spiked Samples – Note: This is the protocol the Reisdorph lab used for the ~260 endogenous food compounds. Final concentrations may vary in the future. Spike 20 ng of each standard for every 10 mg of food. The lowest concentration of compounds in a standard mix can be 0.166 ng/uL. If the concentration of standards is 0.166 ng/uL then the food must be extracted 100% in the standard solution. If you receive 6 mixes of standards at 1 ng/uL concentration and pool all 6 equally your final concentration of all standards will be 0.166 ng/uL.
  6. Pre-chill Tissue-Lyser sample holder at 4˚C in the refrigerator for at least 10 minutes and place samples on Wet-Ice.
  7. Bead homogenize the samples in the tissue-lyser for 2 minutes at 50 hz.
  8. Repeat homogenization step “g” 2 more times, chilling samples and sample holder at 4˚C for 5 minutes between each bead homogenization to avoid sample heating.
  9. Centrifuge samples at 18,000 g at RT or 4˚C for 10 min to pellet food. If the standards are not soluble at 4˚C then centrifuge at room temperature.
  10. Aliquot 120 uL of supernatant to be used for Captiva SPE cleanup. Note: if NOT using Captiva, then a methanol crash can be performed (see Methanol Crash Protocol below)

Captiva EMR-Lipid, 1mL 40 mg SPE protocol

Protocol based on Captiva EMR-Lipid instruction manual.

  1. PRE-WASH CAPTIVA COLUMNS
    This step is essential for removing contaminants that result in ion suppression and can affect chromatography. This step is not included in the Agilent Captiva EMR-Lipid protocol.
  • Add 120 uL of 4:1 Methanol:Water + 480 uL of crash solvent to Captiva column
  • Flow solvent through Captiva column to waste at a rate no quicker than 1 drop every 3 to 5 seconds using a positive pressure manifold or vacuum manifold if positive pressure manifold is not available. It is OK to flow slower than 1 drop every 3-5 seconds but NOT faster.
    Note: It is suggested that for positive pressure manifold you start at 2 psi and slowly increase pressure depending on the flow through rates observed in all samples.
    For all steps, make sure the column is uniformly wetted.
  1. After the solvent has passed through the column, completely dry the Captiva resin by continuing to vacuum or flow nitrogen (positive pressure) through the Captiva column for approximately 5-10 minutes. This time estimate is for a positive pressure manifold.
  2. Add 120 uL aliquot of supernatant from homogenized food sample to the pre-washed and dried Captiva column.
  3. Add 480 uL of crash solvent to the Captiva column.
  4. Pipette up and down 5 to 10 times to mix the sample.
  5. Flow sample through the Captiva column into an MRQ Autosampler vial or 1.5mL microfuge tube at a rate no quicker than 1 drop every 3 to 5 seconds using a positive pressure manifold or vacuum manifold. It is OK to flow slower than 1 drop every 3-5 seconds but NOT faster.
  6. Dry down flow-through collected in step “f” under nitrogen at 35°C. (~30 to 45 minutes)
  7. Re-suspend the sample in 40 uL of 80% Methanol in water and store at -75°C for LC-MS analysis
  8. Inject 2 uL of sample for LCMS which corresponds to 1 ng of standards on column and 0.5 mg of food. This is based on initial protocol used by Reisdorph lab.

Methanol Crash Protocol – TO BE PERFORMED ONLY IF NOT USING CAPTIVA

  1. Transfer 120 uL aliquot of supernatant from homogenized and extracted food sample to a 1.5 mL microfuge tube and store in the -20˚C freezer for 1 hour.
  2. Remove sample from freezer and centrifuge sample at 18,000 g for 15 min at 4C to pellet any precipitated protein.
  3. Aliquot 105 uL of sample carefully so as not to disturb protein pellet to a an appropriately labeled MRQ Autosampler vial or Autosampler vial compatible with user LC/MS system.
  4. Dry down sample under nitrogen at RT ( ~30 min)
  5. Re-suspend in 35 uL of 80% methanol.
  6. Inject 2 uL of sample for LCMS which corresponds to 1 ng of standards on column and 0.5 mg of food. This is based on initial protocol used by Reisdorph lab.
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